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Adenovirus-transfected primary cells and methods of pathway mappingRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test StripAdenovirus-transfected primary cells and methods of pathway mapping description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070166696, Adenovirus-transfected primary cells and methods of pathway mapping. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] The invention also relates to the field of pathway mapping, and in particular to pathway mapping immunocytes that are not immortal cell lines. The invention further relates to the field of modulating the expression of intracellular signaling pathways. [0003] 2. Description of Related Art [0004] An intracellular signaling pathway is a set of genes whose expression is controlled by a cascade of gene-to-gene interactions begun by an initial stimulus. In other words, the initial stimulus of an intracellular signaling pathway controls the expression of each gene on the pathway either directly or indirectly through another gene on the pathway. Known signaling pathways are involved in a wide range of biological activity including immune response, development, mitosis, and inflammation, and include, but are not limited to, the mitogen-activated protein kinase (pathways, the protein kinase A ("PKA") signaling pathway, the protein kinase C ("PKC") signaling pathway, the Type I interferon signaling pathway, the calcineurin ("CaN") signaling pathway, the cis-acting nuclear factor of .kappa.B cells ("NF.kappa.B") signaling pathway, and the I.kappa.B kinase ("IKK") signaling pathway. Although defects in signaling pathways are implicated in a wide range of disease states, in many cases little is understood about a signaling pathway between the initial stimulus and the ultimate cellular response. [0005] Pathway mapping refers generally to elucidating the steps between a pathway's initial stimulus and ultimate responses. For the purposes of this invention, "pathway mapping" refers to the determination of whether a candidate cis-acting regulatory element is controlled, either directly or indirectly, by a given signaling pathway. A known method of pathway mapping employs host cells from a cultured cell line that are transfected with a DNA plasmid containing a reporter gene operatively linked to a regulatory element to be tested. The use of reporter genes is well established in the art. Kain, SR, and Ganguly, S, Overview of Genetic Reporter Systems, Unit 9.6 in Current Protocols in Molecular Biology, Ed. Ausubel, F M, et al., (Wiley and Sons, NY, 1995). [0006] Transfection refers to the introduction of foreign DNA into a host cell, such as a cultured mammalian cell. Transfection has proved to be a powerful tool for analyzing the function, regulation, and interaction of genes and their respective gene products. Procedures for transfecting cells are well-established in the art. Chemical and physical methods of transfection include DEAEdextran (Fox R M, et al, Biochemistry October 4; 16(20): 4470-7, PMID 911769 (1977)), calcium phosphate (Pear, W. S., Nolan, G. P., Scott, M. L., & Baltimore, D. Proc. Natl. Acad. Sci. USA 90, 8392 (1993)), liposome-mediated transfection (Zhang W W, et al., Biotechniques November; 15(5):868-72 (1993)), micro-injection (Colbere-Garapin F, and Garapin A C, Dev Biol Stand; 55:267-71, PMID: 6329857 (1983)), and electroporation (Neumann E and Kakorin S, Technol Cancer Res Treat. October; 1(5):32940(2002)). The use of recombinant adenovirus vectors for transfection is also well established in the art. Berkner, K L and Sharp, Pa. Nucelic Acids Res. 11:6003-6020 (1983); Berkner, K and Sharp, Pa. BioTechniques 6:616-629 (1988); Bett, A J, Haddara, W, Prevec, L and Graham, Fla. PNAS USA 91:8802-8806 (1994); Hitt, M, Addison, C L, and Graham, F L Adv. Pharmacol. 40:137-206 (1997). Recombinant adenoviruses have been used to transfect, inter alia, cardiac tissue, Ardehali, A. J. Thor. Cardiovas. Surg. 111:246-252 (1996), and lymphocytes, Leon, R P, Hedlund, T, Meech, S J, Li, S, Schaack, J, Hunger, S P, Duke, R C, and DeGregori, J PNAS USA 95:13159-13164 (1998). Human adenoviruses are double-stranded DNA viruses which enter cells by receptor-mediated endocytosis. These viruses have been considered well suited for gene transfer because they are easy to grow and manipulate and they exhibit a broad host range in vivo and in vitro. Adenoviruses are able to infect quiescent as well as replicating target cells and persist extrachromosomally, rather than integrating into the host genome. Recombinant adenoviruses can accommodate relatively large segments of foreign DNA (about 7 kb), and have the advantage of a high titer virus production. [0007] Known vectors for reporter constructs used for pathway mapping include the pCRE-d2EGFP plasmid, available commercially from BD Biosciences Clontech. This plasmid contains a cyclic-AMP response element ("CRE") operatively linked to a green fluorescent protein ("GFP") reporter gene, and can be used to measure activity on pathways such as the Jun N-terminal kinase ("JNK") signaling pathway, the p38 MAPK signaling pathway, and the protein kinase A ("PKA") signaling pathway. This plasmid uses the pUC origin of replication for propagation in E. coli, and a viral f1 origin for production of single stranded DNA. This plasmid transfects in eukaryotic cells at a much lower efficiency than adenovirus-based vectors, and are only efficient enough to be used on cultured eukaryotic cells lines and not primary eukaryotic cell cultures. Because known vectors for pathway mapping of immunocytes are not efficient enough to be practical for use on primary immunocytes, pathway mapping using methods of the prior art can currently only be performed on cultured cell lines. However, immortalized cultured cell lines tend to have a significantly different patterns of protein expression than their non-immortalized counterparts. It is desirable to be able to perform pathway mapping on primary host cells of higher organisms without unnecessarily disrupting their cells' normal pattern of expression. It is especially desirable to be able to perform pathway mapping on primary immunocytes without having to transform them into immortal cell lines. BRIEF SUMMARY OF THE INVENTION [0008] In one aspect, the invention relates to a method for determining whether a stimulus known to modulate expression of a signaling pathway in an immunocyte is capable of modulating expression of a candidate cis-element having the steps of transfecting the immunocyte with a recombinant adenovirus having a reporter gene operatively linked to the candidate cis-element; measuring a base level of reporter gene activity; applying the stimulus to the immunocyte; and measuring reporter gene activity in response to said stimulus. In a further aspect, the stimulus is modulating expression of a regulatory protein. In yet a further aspect, the method further comprises co-transfection with an expression system for the regulatory protein. In an alternate aspect, the stimulus is introducing a candidate regulatory compound. In a further aspect, the reporter gene is luciferase, green fluorescent protein ("GFP"), .beta.-galactosidase ("GAL"), chloramphenicol acetyltransferase ("CAT"), or a supressor gene such as I.kappa.Bsd. In another aspect, the cis-element is related to inflammation. In another aspect, the cis-element is AP-1, CRE, ISRE, NFAT, NF.kappa.B, or SRE. In another aspect, the immunocyte is a macrophage, CD4.sup.+ T cell, or immature dendritic cell. [0009] In one aspect, the invention relates to a method for inhibiting expression of a signaling pathway in an immunocyte having the steps of transfecting the immunocyte with a recombinant adenovirus containing a suppressor gene operatively linked to a cis-element belonging to the signaling pathway; and inducing expression of the suppressor gene. In a further aspect, the signaling pathway is the NF.kappa.B signaling pathway. In yet a further aspect, the suppressor gene is I.kappa.Bsd. In a further aspect, the immunocyte is a macrophage, CD4+ T cell, or an immature dendritic cell. BRIEF DESCRIPTION OF THE DRAWINGS [0010] FIG. 1A depicts macrophages transfected with a recombinant adenovirus capable of expressing green fluorescent protein ("AdV:GFP"). [0011] FIG. 1B depicts dendritic cells transfected with a recombinant adenovirus capable of expressing green fluorescent protein ("AdV:GFP"). [0012] FIG. 1C depicts green fluorescent protein expression in T lymphocytes. [0013] FIG. 1D depicts green fluorescent protein expression in B lymphocytes. [0014] FIG. 2 depicts cell two separate cell cultures, one transfected by a recombinant adenovirus capable of expressing a superdominant mutant of I.kappa.B ("Adv:I.kappa.Bsd") and the other transfected by a recombinant adenovirus capable of expressing green fluorescent protein ("AdV:GFP"), each depicted in phase contrast and propidium iodide staining. [0015] FIG. 3A is a schematic diagram depicting a superdominant mutant of I.kappa.B ("I.kappa.Bsd") blocking the NF.kappa.B signaling pathway. [0016] FIG. 3B is a chart depicting the effects of inhibition of NF.kappa.B activation on inflammatory cytokine expression during dendritic cell maturation. [0017] FIG. 4 is a schematic depicting a recombinant adenovirus-based promoter/reporter system. [0018] FIG. 5A is a chart depicting the activation of CRE cis-elements in CD4.sup.+ T cells upon stimulation. [0019] FIG. 5B is a chart depicting the activation of AP-1 cis-elements in CD4.sup.+ T cells upon stimulation. [0020] FIG. 5C is a chart depicting the activation of NF.kappa.B cis-elements in CD4.sup.+ T cells upon stimulation. [0021] FIG. 5D is a chart depicting the activation of ISRE cis-elements in CD4.sup.+ T cells upon stimulation. Continue reading about Adenovirus-transfected primary cells and methods of pathway mapping... 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